Wendy J. Smith

Metagenomics of the svalbard reindeer rumen microbiome reveals abundance of polysaccharide utilization loci

Lignocellulosic biomass remains a largely untapped source of renewable energy predominantly due to its recalcitrance and an incomplete understanding of how this is overcome in nature. We present here a compositional and comparative analysis of metagenomic data pertaining to a natural biomass-converting ecosystem adapted to austere arctic nutritional conditions, namely the rumen microbiome of Svalbard reindeer (Rangifer tarandus platyrhynchus). Community analysis showed that deeply-branched cellulolytic lineages affiliated to the Bacteroidetes and Firmicutes are dominant, whilst sequence binning methods facilitated the assemblage of metagenomic sequence for a dominant and novel Bacteroidales clade (SRM-1). Analysis of unassembled metagenomic sequence as well as metabolic reconstruction of SRM-1 revealed the presence of multiple polysaccharide utilization loci-like systems (PULs) as well as members of more than 20 glycoside hydrolase and other carbohydrate-active enzyme families targeting various polysaccharides including cellulose, xylan and pectin. Functional screening of cloned metagenome fragments revealed high cellulolytic activity and an abundance of PULs that are rich in endoglucanases (GH5) but devoid of other common enzymes thought to be involved in cellulose degradation. Combining these results with known and partly re-evaluated metagenomic data strongly indicates that much like the human distal gut, the digestive system of herbivores harbours high numbers of deeply branched and as-yet uncultured members of the Bacteroidetes that depend on PUL-like systems for plant biomass degradation.

16S rDNA sequencing of Ruminococcus albus and Ruminococcus flavefaciens : design of a signature probe and its application in adult sheep

The ruminococci are an important group of fibrolytic bacteria inhabiting the rumen. Seventeen strains of presumptively identified Ruminococcus were evaluated by a combination of nearly complete and partial 16S rDNA sequence that identified all strains as either Ruminococcus albus or Ruminococcus flavefaciens. All sequences fell into cluster IV of the clostridia, while other species of ruminococci (e.g. Ruminococcus obeum, Ruminococcus gnavus, Ruminococcus lactaris) fall into cluster XIVa of the clostridia. Ruminococcus cluster IV sequences were used to design a 16S rRNA oligonucleotide probe to assess the relative abundance of target populations in a stable ruminal environment. A stable population (animals fed eight times per day) was established in sheep so that statistically robust comparisons could be made in the absence of variation due to diurnal rumen fluctuations. The steady state populations were sampled six times over a 24 d period and direct microscopic counts (DC), total culturable counts (TCC), and total cellulolytic counts (CEL) were determined. DC and culturable data (TCC and CEL) were compared with relative abundance estimates of Ruminococcus IV and Fibrobacter succinogenes. A combination of the Ruminococcus and F. succinogenes probes accounted for 4.0% of the bacterial population and cellulolytic bacteria (measured by most-probable numbers) were 5.2% of the total culturable count. These data suggest that a major portion of the Ruminococcus and Fibrobacter diversity has been cultured and is represented by available sequences. Steady state populations were measured over several days in three sheep and an estimate of variation in DC, TCC, CEL and 16S-based data were obtained. These variance estimates could be used to determine the theoretical sample sizes required to obtain statistically significant differences under different experimental conditions.

Isolation of Succinivibrionaceae Implicated in Low Methane Emissions from Tammar Wallabies

Metagenome sequence predicted the culture conditions required for successful isolation of a marsupial gut bacterium. The Tammar wallaby (Macropus eugenii) harbors unique gut bacteria and produces only one-fifth the amount of methane produced by ruminants per unit of digestible energy intake. We have isolated a dominant bacterial species (WG-1) from the wallaby microbiota affiliated with the family Succinivibrionaceae and implicated in lower methane emissions from starch-containing diets. This was achieved by using a partial reconstruction of the bacterium’s metabolism from binned metagenomic data (nitrogen and carbohydrate utilization pathways and antibiotic resistance) to devise cultivation-based strategies that produced axenic WG-1 cultures. Pure-culture studies confirm that the bacterium is capnophilic and produces succinate, further explaining a microbiological basis for lower methane emissions from macropodids. This knowledge also provides new strategic targets for redirecting fermentation and reducing methane production in livestock.

Use of 16S-rRNA based techniques to investigate the ecological succession of microbial populations in the immature lamb rumen: Tracking of a specific strain of inoculated Ruminococcus and interactions with other microbial populations in vivo

A bstractThe establishment of microorganisms in the rumen is a critical step if rumen manipulation is to be accomplished by use of microbial inoculants. Microbial populations in the maturing rumen undergo successional changes and, while in a state of flux, provide a possible opportunity for the introduction of specific strains of bacteria. While the rumen of the young lamb was maturing, we measured changes in several microbial populations with 16S-rRNA specific oligonucleotides: Rumincoccus, Fibrobacter, eukaryotes, Gram-positive bacteria, the Bacteroides–Porphromonas–Prevotella group, and anaerobic rumen fungi. In this study we repeatedly dosed 15 lambs with approximately 3.4 × 108 to 0.8 × 109Ruminococcus cells dose-1, twice a week, for 7 wk from 23 d to 63 d of age. Of the five Ruminococcus strains dosed (R. albus SY3 and AR67, and R. flavefaciens Y1, LP9155, and AR72) the most specific primers (based on 16S rDNA) were obtained for strain SY3. There was an increase in the eukaryotic population during dosing, and it was hypothesized that protozoal predation contributed to the disappearance of strain SY3. At the end of dosing PCR amplification showed that SY3 were approximately 109 cells ml-1, but decreased to below the detection limit of the PCR system (8.6 × 104 ml-1) within 28 d postdosing. These experiments showed that fibrolytic populations increased significantly (P < 0.1) above the controls during the dosing period and were elevated for several days postdosing. This suggests that dosing of highly fibrolytic bacteria makes more of the fiber available to other organisms able to degrade fiber, and in so doing increases the overall fibrolytic activity of the rumen. Examination of the succession of gram-positive bacteria and the Bacteroides–Porphromonas–Prevotella group showed a decline in relative abundance as the lambs matured.

Repeated ruminal dosing of Ruminococcus spp. does not result in persistence, but changes in other microbial populations occur that can be measured with quantitative 16S-rRNA-based probes.

Digestibility of fibre in ruminants may be improved by the introduction of highly fibrolytic strains of ruminal bacteria. This approach may be feasible if, for example, strains of Ruminococcus that are significantly more fibrolytic than the normal population of Ruminococcus are used for inoculation purposes. Introduced strains of bacteria, irrespective of ecosystem, often decline after inoculation, and in this study, highly fibrolytic strains of Ruminococcus were continuously dosed to ensure that measurements of fibre digestion were made in the presence of significant numbers of the introduced bacteria. During dosing the total culturable count increased significantly (P<0.05), but declined post-dosing. The level of dosed Ruminococcus, and total Ruminococcus, Fibrobacter succinogenes and eukaryotes measured by 16S rRNA probes increased significantly (P<0.05) during the dosing period, but also declined post-dosing. When in vitro nylon bag digestibility, feed intake or whole-tract digestibility was measured, no improvement could be measured.

Diversity of Ruminococcus strains: a survey of genetic polymorphisms and plant digestibility

Twenty‐three strains of Ruminococcus isolated from ruminants were assessed for digestive ability on different plants and purified cellulose. Genetic diversity was assessed by ERIC, REP and 16–23S rDNA spacer polymorphisms. All ruminococci could be typed by ERIC, REP or 16–23S rDNA spacer, but all three typing methods had to be used in concert to differentiate closely related strains. Digestibility of lucerne (Medicago sativa), rhodes grass (Chloris gayana) and spear grass (Heteropogon contortus) were assessed. Dry matter (DM) digestibility was highly correlated (> 0·93) with neutral detergent fibre (NDF) digestibility, but cellulose disc digestibility was a poor indicator of DM and NDF digestibility. Studies demonstrate the wide variation in ability of ruminococci to digest forages, and some recently isolated strains (Y1, LP‐9155, AR67, AR71 and AR72) were superior to reference strains (FD‐1 and Ra8). Multivariate analysis showed that groupings derived from genotyping data closely resembled those determined by digestibility data. This study indicated that ruminococci are diverse in digestive ability and genotype, and this diversity suggests that there may be highly fibrolytic strains in nature that could be utilized for animal production.

A genomics-informed, SNP association study reveals FBLN1 and FABP4 as contributing to resistance to fleece rot in Australian Merino sheep

BackgroundFleece rot (FR) and body-strike of Merino sheep by the sheep blowfly Lucilia cuprina are major problems for the Australian wool industry, causing significant losses as a result of increased management costs coupled with reduced wool productivity and quality. In addition to direct effects on fleece quality, fleece rot is a major predisposing factor to blowfly strike on the body of sheep. In order to investigate the genetic drivers of resistance to fleece rot, we constructed a combined ovine-bovine cDNA microarray of almost 12,000 probes including 6,125 skin expressed sequence tags and 5,760 anonymous clones obtained from skin subtracted libraries derived from fleece rot resistant and susceptible animals. This microarray platform was used to profile the gene expression changes between skin samples of six resistant and six susceptible animals taken immediately before, during and after FR induction. Mixed-model equations were employed to normalize the data and 155 genes were found to be differentially expressed (DE). Ten DE genes were selected for validation using real-time PCR on independent skin samples. The genomic regions of a further 5 DE genes were surveyed to identify single nucleotide polymorphisms (SNP) that were genotyped across three populations for their associations with fleece rot resistance.ResultsThe majority of the DE genes originated from the fleece rot subtracted libraries and over-representing gene ontology terms included defense response to bacterium and epidermis development, indicating a role of these processes in modulating the sheeps response to fleece rot. We focused on genes that contribute to the physical barrier function of skin, including keratins, collagens, fibulin and lipid proteins, to identify SNPs that were associated to fleece rot scores.ConclusionsWe identified FBLN1 (fibulin) and FABP4 (fatty acid binding protein 4) as key factors in sheeps resistance to fleece rot. Validation of these markers in other populations could lead to vital tests for marker assisted selection that will ultimately increase the natural fleece rot resistance of Merino sheep.

Expression of a modified Neocallimastix patriciarum xylanase in Butyrivibrio fibrisolvens digests more fibre but cannot effectively compete with highly fibrolytic bacteria in the rumen

Aims: This study investigated the competitive abilities of two Neocallimastix patriciarum‐derived xylanases constructs in Butyrivibrio fibrisolvens H17c (xynA and pUMSX) and their ability to compete in vivo.

Isolation of genetically tractable most-wanted bacteria by metaparental mating

Metagenomics has rapidly advanced our inventory and appreciation of the genetic potential inherent to the gut microbiome. However it is widely accepted that two key constraints to further genetic dissection of the gut microbiota and host-microbe interactions have been our inability to recover new isolates from the human gut, and the paucity of genetically tractable gut microbes. To address this challenge we developed a modular RP4 mobilisable recombinant vector system and an approach termed metaparental mating to support the rapid and directed isolation of genetically tractable fastidious gut bacteria. Using this approach we isolated transconjugants affiliated with Clostridium cluster IV (Faecalibacterium and Oscillibacter spp.), Clostridium cluster XI (Anaerococcus) and Clostridium XIVa (Blautia spp.) and group 2 ruminococci amongst others, and demonstrated that the recombinant vectors were stably maintained in their recipient hosts. By a similar approach we constructed fluorescently labelled bacterial transconjugants affiliated with Clostridium cluster IV (including Flavonifractor and Pseudoflavonifractor spp.), Clostridium XIVa (Blautia spp.) and Clostridium cluster XVIII (Clostridium ramosum) that expressed a flavin mononucleotide-based reporter gene (evoglow-C-Bs2). Our approach will advance the integration of bacterial genetics with metagenomics and realize new directions to support a more mechanistic dissection of host-microbe associations relevant to human health and disease.

Gene expression profiling of ovine skin and wool follicle development using a combined ovine–bovine skin cDNA microarray

Low fibre diameter and high fleece weight are important determinants of the economic value of the Merino fleece. The combination of these traits is found in Merino sheep with high follicle densities resulting from a high secondary to primary follicle ratio. Morphological stages in the development of primary and secondary follicles of fetal sheep skin have been well described. We have used gene expression profiling of fetal skin to identify genes that may be important in controlling these follicle developmental processes. A combined ovine (2.3 K) and bovine (6.14 K) cDNA microarray of 2 fetal and 1 adult stage skin tissues was constructed to compare gene expression levels between fetal day 82, day 105, day 120 and adult sheep skin developmental stages. The transcript profile resulted in 238 differentially expressed array elements relative to the adult expression, which represented 132 unique genes. These clustered into 50 up- and 82 down-regulated genes and distinct gene ontologies including structural constituents, phosphate transport, signal transduction and organogenesis. Northern blot analysis of 2 selected genes, S100A7LI and TAGLN, validated the microarray results. This list of genes contains candidates of interest for further investigation into the molecular control of wool follicle development.